Frequent and extensive Cas9-directed genomic damage
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB25982 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR2512074_subreads.fastq.gz | Fastqsanger.gz | |||
| ERR2512075_subreads.fastq.gz | Fastqsanger.gz | |||
| ERR2512076_subreads.fastq.gz | Fastqsanger.gz | |||
| ERR2512077_subreads.fastq.gz | Fastqsanger.gz | |||
| ERR2512078_subreads.fastq.gz | Fastqsanger.gz |
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Project description:CRISPR-Cas is an RNA-based defense system that enables prokaryotes to recognize invading foreign DNA by cognate crRNA guides and destroy it by CRISPR-associated Cas nucleases 1,2 . Elucidation of the interference mechanism of the Streptococcus pyogenes Type II CRISPR- Cas9 system has allowed for the successful repurposing of SpCas9 as a generic genome editing tool, with great promise for human gene therapy 3 . However, especially for therapeutic applications, some caution seems appropriate, because Cas9 systems from some human pathogens may induce a cytotoxic response via an unknown mechanism 4 . Here we show that when released in human cells, Cas9 nucleases from the pathogenic bacteria Campylobacter jejuni and S. pyogenes have the potential to cause severe DNA damage. In the absence of a CRISPR RNA guide, native Cas9 nucleases from both pathogens enter the host nucleus, where their presence leads to promiscuous double stranded DNA breaks (DSBs) and induction of cell death. DSB induction can be reduced to background levels either by saturation of CjCas9 and SpCas9 with crRNA guides or by inactivating their nuclease activity. Our results demonstrate that guide-free Cas9 of bacterial pathogens might play an important role in pathogenicity. Furthermore, we propose that saturating Cas9 with appropriate guide RNAs is crucial for efficient and safe therapeutic applications.
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Project description:Based on the hypothesis that, enhancing the local concentration of donor oligos could increase the correction rates, we generated and tested novel CRISPR-Cas9 systems, in which the DNA repair template is covalently conjugated to Cas9 (RNPD system). To validate our results from the HEK293T reporter cells, we here tested our approach at different endogenous genomic loci and in different cell types. We first targeted the human beta globin (HBB) locus in the K562 cell line, and analyzed correction- and editing frequencies using next generation sequencing (NGS). Next we targeted the Rosa26 and proprotein convertase subtilisin/kexin type 9 (Pcsk9) locus in mouse embryonic stem cells (mESCs). Here, RNPD system was always compared to Cas9 SNAP-tag fusion proteins with uncoupled donor oligos. To also directly compare the engineered RNPD system to the classical CRISPR-Cas9 system, we performed experiments where we used wild-type Cas9 with the uncoupled donor oligos as a control. We therefore targeted the fluorescent reporter locus as well as the endogenous loci HBB, empty spiracles homeobox 1 (EMX1), and C-X-C chemokine receptor type 4 (CXCR4) in HEK293T cells. Finally, we performed the analysis of three computationally predicted off-target sites of the reporter locus.
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Project description:The evolutionarily conserved POT1 protein binds the single stranded G-rich telomeric DNA and has been implicated in telomeric DNA maintenance and the suppression of DNA damage checkpoint signaling. Here, we explore human POT1 function through genetics and proteomics discovering that the complete absence of POT1 leads to severe telomere maintenance defects that had not been anticipated from previous depletion studies. We determine the telomeric proteome upon POT1-loss by implementing an improved telomeric chromatin isolation protocol. Using quantitative proteomics by tandem mass tags (TMT) we identified a large set of proteins involved in nucleic acid metabolism that engage with telomeres upon loss of POT1. Inactivation of the homology directed repair machinery suppresses POT1-loss mediated telomeric DNA defects. Our results unravel as major function of human POT1 the suppression of telomere instability induced by homology directed repair.
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Project description:Guide-free Cas9 from pathogenic Campylobacter jejuni bacteria causes severe damage to DNA
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